Construction And Therapeutic Analysis Of Bi-Specific Antibodies Targeting GPRC5D And CD3

Multiple myeloma(MM)is a fatal disease caused by the malignant proliferation of plasma cell clones,which accounts for 10%of all blood tumors.In recent years,the rapid development of tumor immunotherapy technologies,represented by antibody drug conjugates(ADCs),bispecific antibodies and chimeric antigen receptor T cells(CAR T),has brought new hope to MM patients.Due to the heterogeneity of MM genetics and phenotypes,many targets are expressed by MM cells,such as BCMA,CD38 and CD 13 8.These targets cause tumor escape from the surface of tumor cells or falling off,which is an important reason for MM to relapse or difficult to treat.Therefore,it is urgent to develop antibodies that binding to different antigen or different epitope of the same antigen to effectively avoid the reduction or drug resistance caused by antigen mutations or loss,and enhance the long-term persistence of drugs.GPRC5D(G protein-coupled receptor class C group 5 member D)is a member of the G-protein-coupled receptor superfamily RAIG1(retinoic acid inducible gene 1).GPRC5D is not only stably expressed on the cell membrane surface without shedding,but is also not expresses in normal cells except for hair follicles and is only significantly increased in MM cells.This is a good target for the treatment of MM.However,the development of antibodies against GPRC5D is severely limited by its complex seventransmembrane structure and small extracellular structural domain,which is difficult to purify.Therefore,this paper attempted to determine the specificity of natural surface antibodies to GPRC5D by using GPRC5D expression plasmids and expression cell lines as immunogens in combination with animal immunization and phage display techniques.In addition,it was applied together with CD3 antibody for the construction and functional evaluation of bispecific antibodies,providing new ideas for immunotherapy of MM.In this study,Balb/C mice were immunized with 293T-Hu.GPRC5D cells and GPRC5D plasmid PLVX-GPRC5D,respectively.The antibody titers in the sera of the immunized mice were detected by FACS.The results showed that no antibody binding to GPRC5D was detected in serum of mice immunized with the plasmid,but serum titer of mice immunized with the cells reached a titer of 1:100,000.Then,a phage antibody library was constructed from spleen cells of immunized mice(1.16×109 library size,clonal positive rate>95%).GPRC5D-negative and overexpressing 293T cells were then applied for negative and triple-positive panning selection.Nearly 150 clones with higher binding activity than the positive control antibodies were screened.Finally,six antibody sequences were identified by sequencing.After expression and purification,five antibodies with higher binding activity than the positive control ET150-8BMK to NCI-H929 cells were obtained.After further homology analysis and comparison,xw.HTS037 and xw.HTS0375 were finally selected for subsequent humanization modification.In addition,we also tested the tissue cross-reactivity of the humanized antibodies with a variety of normal cells,and the results showed that humanized antibody zw.HTS0370Z22 and zw.HTS0375Z56 didn’t bind to B cells,T cells,CD14+monocytes and CD11b+cells,showing high cell specificity.In contrast,ET150-8BMK bound to CD11b+and CD14+cells to some extent.The results of antibody affinity determination showed that zx.TS0375Z56 and zw.HTS0370Z22 have a higher binding affinity with human GPRC5D protein.Subsequently,we used the exocytic segment of GPRC5A that did not bind to the humanized antibody to replace the corresponding exocytic segment of GPRC5D at the same position,and constructed a series of GPRC5D mutant cell lines.Then,a series of binding experiments of GPRC5D mutants with antibodies indicated that zw.HTS0375Z56 and zw.HTS0370Z22 might bind to ECD3 and ECD4 extracellular domains at the C-terminal of the GPRC5D protein,which was different from the reported epitope binding to GPRC5D antibody.In order to further explore the potential of GPRC5D humanized antibodies in MM therapy,GPRC5D humanized monoclonal antibody was developed.Antibodies of zw.HTS0370Z22 as well as zw.HTS0375Z56 and CD3 were constructed as DouBody and CrossMab IgG bispecific antibodies,respectively.The GPRC5D protein and CD3 protein prepared from VLP were detected by ELISA,and all four antibodies were found to bind to GPRC5D protein with better binding activity than the positive control GC5B596D.However,370Z22KC and 375Z56KC showed significantly better binding activity and affinity to GPRC5D than 370Z22D and 375Z56D.Among them,375Z56KC showed the best binding activity against the tumor cell line NCI-H929.375Z56KC bispecific antibody has the highest single-arm affinity binding to human GPRC5D protein,about 6 times the single-arm affinity of control bispecific antibody GC5B596 developed by Johnson&Johnson company.Subsequently,it was demonstrated that 375Z56KC showed the highest anti-tumor activity when the bispecific antibodies were evaluated in vitro using LDH release assays in PBMC and tumor cell culture systems.Moreover,the activation of NFAT transcription activity was most pronounced in Jurkat-NFAT-Luc cells.Therefore,375Z56KC was selected for subsequent in vivo animal experiments.Firstly,2×106/mouse of NCI-H929 cells were inoculated subcutaneously into mice.One day after inoculation,2×106/mouse healthy human PBMC were injected into the mice.When tumor growth reached 100 mm3,the bispecific antibodies were dosed at 1.5μg/mouse or 6μg/mouse(two injections per week for a total of five).The results showed that both positive control group and 375Z56KC group could significantly inhibit tumor growth compared with Vehicle control group.While the tumor inhibition effect of 375Z56KC group was significantly higher than that of positive control group.Only 1.5μg/mouse was required to completely inhibit tumor growth.Meanwhile,the T cell ratio,CD4+T cells,CD8+T cells and serum IFN-γ content in the 375Z56KC dual anti-treatment group were significantly higher than those in the Vehicle group and the GC5B596D positive control group.Consistenting with the flow analysis,immunofluorescence analysis of tumor tissues also showed a significant increase in the number of CD4+T cells and CD8+T cells in the 375Z56KC bispecific antibody treated group,indicating that 375Z56KC bispecific antibody has a good tumor immunotherapeutic effect.Hematoxylin and eosin staining analysis of vital organs in mice showed no significant organ damage in the positive control and 375Z56KC bispecific antibody treatment group.At the same dose of 24 μg/mouse(twice a week,6 times in total),375Z56KC bispecific antibody significantly inhibited the growth of mouse MM.1S and was significantly more effective than the positive control group treatment,suggesting that 375Z56KC bispecific antibody had good binding activity and killing effect on a variety of MM cell lines both in vivo and in vivo.Bispecific antibody has become a hot topic in the field of tumor immunotherapy because of its great breakthrough in clinical treatment.In the paper,a novel monoclonal antibody against GPRC5D was screened by using immune cell and cell-based phage antibody library panning selection.On the basis of humanization,specific double antibodies with different structural features were constructed using CD3-specific antibodies.The antitumor function were evaluated in vivo and in vitro comprehensively.The results showed that the bispecific antibody 375Z56KC constructed in this paper not only had good anti-tumor effects in vivo and in vitro,effectively enhancing the antitumor T cell-mediated immune response,but also had significantly better effects than the positive control bispecific antibody GC5B596D developed by Johnson&Johnson company.This study provides a new tool as well as an idea for the immunotherapy of MM tumor.